Device and method for determining the concentration of fluorophores in a sample

ABSTRACT

A device ( 10 ) and a method for analyzing a sample ( 16 ) containing fluorophores use a light source ( 12 ) emitting light (λ ex ) onto the sample ( 16 ), and onto a fluorescence standard ( 14 ). The fluorophores of the sample ( 16 ), given an immission of light of a first wavelength (λ ex1 ), have a first excitation efficiency and, given an immission of light of a second wavelength (λ ex2 ), have a second excitation efficiency. The fluorescence standard ( 14 ), given the same immissions of light, has a third excitation efficiency and, a fourth excitation efficiency. An optical element ( 20 ) which is arranged between the light source ( 12 ) and the sample ( 16 ) and/or ( 12 ) the fluorescence standard ( 14 ) adapts, due to its optical property, a first difference between the first excitation efficiency and the second excitation efficiency and a second difference between the third excitation efficiency and the fourth excitation efficiency to each other.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of German patent application No. 102011 002 080.2, filed on Apr. 15, 2011, the disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to a device for determining the concentration offluorophores in a sample, the sample in particular comprising asubstance marked with fluorophores. Further, the invention relates to acorresponding method.

BACKGROUND OF THE INVENTION

In known devices and methods for the quantitative determination of theconcentration of fluorophores of at least one substance in a sample, thesample is irradiated with light of an excitation wavelength emitted froman excitation light source. The intensity of the fluorescent light of anemission wavelength emitted from the sample is measured by means of adetection element. Such a device and such a method are, for example,known from DE 10 2008 057 115 A1.

In order to guarantee exact measurement results, a reference object, inparticular a so-called fluorescence standard or a suitable opticalelement, is used to calibrate a measured intensity value of thefluorescent light. When irradiated with excitation light of apredetermined wavelength and intensity, the reference object emits lighthaving a known wavelength distribution and/or intensity. The long-termstability of fluorescence standards is often insufficient, whereas theuse of an optical element as a reference object, as known from DE 102008 057 115 A1, guarantees the required long-term stability. Referenceobjects can be used as a calibration standard as well as additionally oralternatively as a quality assurance standard or as a reference standardfor the direct referencing of performed measurements. Referencestandards are often used in so-called scanning systems in which thereference standard and at least one sample are successively irradiatedwith light, and the light that is incident on a detection element andcomes from the reference standard is detected as a first measurementvalue and the light that is incident on the detection element and comesfrom the sample is detected as a second measurement value, wherein bymeans of the ratio of the first measurement value to the secondmeasurement value the concentration of fluorophores in the sample isdetermined. Preferably, the invention, too, can be realized as ascanning system.

From EP 0 237 363 A2, a method and a device for determining thefluorescence of a test sample are known. From U.S. Pat. No. 6,242,114 anoptical composite having fluorescent properties is known, whichcomprises a support layer with fluorescent material, which is opticallycoupled to a selective spectral filter.

In the fluorescence-based analysis of samples, in particular of bodyfluids, for example in the fluorescence-based analysis of sample fluidsapplied to test strips, in known devices absolute values are determinedby a referencing with an internal reference standard. The excitationefficiency of the fluorophore used for analyzing the sample is generallywavelength-dependent so that the sample or the test strip with thesample is irradiated with a narrow-band light having a preset intensityin order to be able to draw conclusions on the number of fluorophoresand thus on the amount of a substance in the sample to which thefluorophores have been attached based on the light emitted from thesample or, respectively, the test strip. A change in the spectrum of thelight source generating the excitation light, in particular a shift inthe wavelength of the light emitted from the light source, partiallyresults in considerable distortions of the measurement result, inparticular a change in wavelength has a very different effect or has aneven opposite effect on the light emitted from the fluorophores and onthe light emitted from the reference object. The wavelength or,respectively, the wavelength spectrum of the light source can, forexample, change due to temperature influences. Further, such a change inwavelength is also possible as a result of so-called spontaneous jumpswhich may occur in the course of the lifetime of the light source. Suchspontaneous jumps, for example, occur in the case of lasers used as alight source. In the case of a change in wavelength as a result oftemperature influences, a compensation can be accomplished with asuitable algorithm by way of a temperature measurement in that thetemperature influences are appropriately taken into account in theevaluation of the measurement values. In the case of the mentionedspontaneous wavelength jumps, such a compensation is not possible sothat there will be faulty measurements.

It is the object of the invention to specify devices and methods fordetermining the concentration of fluorophores in a sample, by which theinfluence on a measurement result due to a changing wavelength of thelight source used for illuminating the sample is reduced.

SUMMARY OF THE INVENTION

This object is solved by a device and method having the features of thepresent invention. Advantageous aspects and developments of theinvention are specified the following description.

By a device and method having the features of the present invention, itis achieved each time that the influence of a change in wavelength ofthe light source on a measurement result to be determined for analyzingthe sample is reduced by the optical element or is completelycompensated for by the optical element. For this, in accordance with thedevice in one form, the optical element can be arranged between thelight source and the sample or between the light source and thereference object. Due to its optical property, the optical elementresults in that a first difference between a first excitation efficiencyof the fluorophores of the sample given an emission of light of a firstwavelength and a second excitation efficiency of the fluorophores of thesample given the emission of light of a second wavelength and a seconddifference between a first excitation efficiency of the fluorophores ofthe fluorescence standard given the emission of light of the firstwavelength and a second excitation efficiency of the fluorophores of thefluorescence standard given an emission of light of the secondwavelength onto the fluorescence standard are brought into approximatecorrespondence with each other. On the other hand, in the deviceaccording to claim 2 the light emitted from the excitation light sourceis emitted onto the sample and emitted onto a monitor diode that isprovided for monitoring the intensity of the light radiation generatedby the light source. By means of such a monitor diode, the intensity ofthe light radiation emitted from the light source can be kept constantvia a control circuit, as this is described, for example, in DE 10 2008057 115 A1.

In particular the ratio of the energy supplied to the sample or,respectively, the fluorophores by the immission of light to the energyused therefrom for the excitation of the fluorophores is referred to asexcitation efficiency.

According to a further aspect of the invention, also a method can beprovided in which by means of an excitation light source excitationlight is emitted onto a substance of a sample containing fluorophores.Further, by means of the excitation light source excitation light isemitted onto a monitor diode. Given an emission of excitation light of afirst wavelength the fluorophores of the sample have a first excitationefficiency and given an emission of excitation light of a secondwavelength they have a second excitation efficiency. The fluorophores ofthe sample emit fluorescent light in the direction of the detectionelement in the case of an immission of excitation light. Further, areference light source for emitting reference light onto the referenceobject and for emitting excitation light onto the monitor diode isprovided. By the optical reference object a part of the reference lightemitted from the reference light source is coupled in the direction of adetection element.

Given an emission of excitation light of a first wavelength, the monitordiode has a first detection sensitivity, and given the emission ofexcitation light of a second wavelength it has a second detectionsensitivity. By means of an optical element arranged between theexcitation light source and the sample and/or between the excitationlight source and the monitor diode a first difference between the firstexcitation efficiency and the second excitation efficiency and a seconddifference between the first detection sensitivity and the seconddetection sensitivity are adapted to each other due to the opticalproperty of the optical element. As a result thereof, the effects of thechange in the detection sensitivity of the monitor diode given a changein wavelength as well as of a change in the excitation efficiency of thefluorophores given a change in wavelength can be reduced or completelycompensated for at least in a wavelength range between the firstwavelength and the second wavelength.

In a development of this further aspect of the invention, as well as ina development of the device using a detection element, at least a partof the fluorescent light of an emission wavelength that is emitted fromthe sample is incident on the detection element which detects acorresponding first measurement value. A part of the reference lightthat is coupled in is incident on the detection element which detects acorresponding second measurement value. The ratio of the firstmeasurement value to the second measurement value is determined, and thenumber of fluorophores of the substance of the sample in a detectionarea is determined taking into account the determined ratio of firstmeasurement value and second measurement value.

It is advantageous when, during a calibration of the device, the opticalreference element couples in the constant part of the reference lightemitted from the reference light source in the direction of thedetection element and a third measurement value corresponding to thepart of the light that is coupled in and that is incident on thedetection element is detected, wherein a fluorescence standard isirradiated with the excitation light emitted from the excitation lightsource, and a fourth measurement value corresponding to the part of thefluorescent light emitted from the fluorescence standard that isincident on the detection element is detected. In this way, an easycalibration of the device by means of a fluorescence standard can beperformed. Further, it is advantageous to use a reemission standard asan optical reference object.

The number of fluorophores in the sample can be determined according tothe following formula:

${FD}_{P} = {\frac{E_{{mes}\; 2}}{E_{s\; 2}} \cdot X}$

whereinFD_(P) is the number of fluorophores of the sample,E_(mes2) is a second measurement value corresponding to the part of thefluorescent light of an emission wavelength coming from the sample andbeing incident on the receiving element,E_(s2) is a first measurement value corresponding to the part of thelight that is coupled in and that is incident on the receiving element,andX is a constant scaling factor which represents a relation between theused optical element (REM) and the fluorescence standard and which isdetermined for the optical element (REM) during the calibration of themeasuring device.

The scaling factor X can be determined according to the followingequation:

$X = {\frac{E_{s\; 1}}{E_{{mes}\; 1}} \cdot {FD}_{FS}}$

wherein

E_(s1) is a third measurement value corresponding to the constant partof the reference light that is emitted from the reference light sourceand is incident on the receiving element, which constant part is coupledin by the optical element in the direction of the receiving elementduring the calibration,

E_(mes1) is a fourth measurement value corresponding to the part of thefluorescent light that is emitted from the fluorescence standard and isincident on the receiving element, andFD_(FS) is the known number of fluorophores of the fluorescence standardin the detection area.

Preferably, the optical path between the reference light source and theoptical reference object that serves to couple in the constant part ofthe reference light emitted from the reference light source in thedirection of the detection element goes through the same opticalelements as the optical path of the excitation light between theexcitation light source and the sample or, respectively, the excitationlight source and the fluorescence standard.

The following embodiments for an advantageous development of theinvention are generally referred to a light source. These developmentscan then be advantageously used in connection with the light source inthe various forms of the invention.

In general, the first wavelength and the second wavelength, the firstexcitation efficiency and second excitation efficiency, the thirdexcitation efficiency and the fourth excitation efficiency, the firstdetection sensitivity and the second detection sensitivity differ fromone another each time.

In an advantageous development of the invention, a detection element fordetecting the light emitted from the sample given an emission of lightof the light source onto the sample and/or for detecting the lightemitted from the fluorescence standard given an emission of light of thelight source onto the fluorescence standard can be provided. In thisconnection, the detection element can determine a feature value of afeature of the light emitted from the sample, such as the brightness orthe intensity of the light radiation, given an emission of light of thelight source onto the sample. Further, the detection element candetermine a feature value of the same feature of the light emitted fromthe fluorescence standard given an emission of light of the light sourceonto the reference object. Due to its optical property, the opticalelement which is arranged between the light source and the sample and/orbetween the light source and the reference object influences, in thecase of a change in wavelength of the light emitted from the lightsource in at least one wavelength range, the light emitted onto thesample and/or the light emitted onto the fluorescence standard and/orthe light emitted from the sample and/or the light emitted from thefluorescence standard such that the detection element determines a firstfeature value that is dependent on the light emitted from the sample anda second feature value that is dependent on the light emitted from thereference object, wherein the change in the difference between the firstfeature value and the second feature value over the wavelength range isreduced or the difference is completely removed by the optical element.In this way, it is achieved that a detection element determinesmeasurement values or, respectively, detection values for analyzing thesample, in particular the intensity of the fluorescence light emittedfrom the fluorophores contained in the sample can be detected easily.For referencing this measurement or for calibrating the device or forquality assurance of the analysis performed by means of the device or,respectively, by means of the method, also the light emitted from thefluorescence standard can be detected by means of the detection elementand a corresponding detection value or, respectively, measurement valuecan be determined. Further, it is advantageous when the detectionelement upon detection of the light emitted from the sample as a resultof an immission of light of the light source onto the sample determinesa detection value by means of which a control unit determines thequantity of the fluorophores in the sample and/or the concentration of asubstance in the sample marked with the fluorophores. In this way, thefluorophore concentration and thus the amount of the substance in thesample marked by means of the fluorophores can be determined easily.

Further, it is advantageous when the optical property of the opticalelement influences the light emitted from the sample upon immission oflight of the light source, the light emitted onto the sample, the lightemitted onto the fluorescence standard, the light emitted onto themonitor diode and/or the light emitted from the fluorescence standardsuch that the first difference is approximated to the second difference,preferably that the second difference is equal to the first difference.In this way, the different excitation efficiencies and detectionsensitivities can at least in part be compensated for by means of theoptical element and thus in an easy manner.

Further, it is advantageous when the optical element adapts the spectralcurve of the excitation efficiency of the fluorophores of the sample andthe spectral curve of the excitation efficiency of the fluorophores ofthe fluorescence standard at least in the wavelength range between thefirst wavelength and the second wavelength. As a result thereof, alsowavelength shifts in the wavelength range between the first wavelengthand the second wavelength are completely or at least in part compensatedfor.

It is particularly advantageous when the first wavelength is the nominalwavelength of the light source and when the second wavelength is awavelength different from the first wavelength, which in particulararises due to temperature influences and/or aging of at least onecomponent of the light source and/or an optical element arranged in thebeam path between light source and sample, between light source andmonitor diode and/or between light source and reference object.According to the invention, such changes in the wavelength between thefirst and the second wavelength can be compensated for easily at leastin part.

The difference between the first wavelength and the second wavelengthcan be in the range between 0.1 nm and 5 nm in the case of common lightsources. As a result thereof, deviations of the wavelength or,respectively, of the wavelength spectrum of the light emitted from thelight source can be compensated for, as they might occur in practice inthe case of common light sources. As a light source in particular alaser light source, an LED light source or a white light source having afilter arrangement for generating light in a narrow-band range can beused. The first wavelength lies in the narrow-band range, preferably inthe middle thereof. This narrow-band range is the nominal wavelengthrange provided for this light source. The second wavelength lies outsidethe middle of this nominal wavelength range, preferably outside thenominal wavelength range. As a result thereof, also a deviation of anominal wavelength range to be generated by the light source arrangementcan be handled by the invention at least such that no or only a littleinfluence on the determined measurement results due to the change inwavelength is to be expected.

Preferably, the light source can emit light successively onto the sampleand onto the fluorescence standard or, respectively, onto the sample andonto the reference object. This is in particular useful in devices inwhich the reference object or the fluorescence standard is not used as areference standard but as a quality assurance standard or as acalibration standard, wherein then the reference object is inserted intothe device or used in the method instead of a sample. As a resultthereof, easy reference measurements between the sample and thereference object or, respectively, the fluorescence standard arepossible so that exact analyses of samples can be performed. Inparticular, the fluorescence standard or, respectively, the referenceobject and at least one sample can be successively illuminated withlight in scanning systems, each time the light that is incident on thedetecting element being detected.

Alternatively, the light source can simultaneously emit light onto thesample and onto the reference object or onto the sample and the monitordiode or onto the sample, the reference object and the monitor diode. Asa result thereof, no adjusting elements have to be provided to emit thelight optionally onto the sample, the monitor diode or the referenceobject.

It is particularly advantageous when the optical element which isarranged between the light source and the sample and/or between thelight source and the reference object or, respectively, between thelight source and the fluorescence standard, is an optical filter,wherein the optical property of the optical element is then thetransmission property of the filter. An optical filter can be producedeasily with a desired optical property. As a result thereof, the opticalproperty of a filter to be produced can be designed and produceddependent on the requirement resulting from the first difference and thesecond difference so that an easy long-time stable compensation of theeffects of a change in wavelength of the light source onto theexcitation sensitivity of the fluorophores, the detection sensitivity ofa monitor diode and/or the emission sensitivity of the reference objectis possible.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the invention result from thefollowing description which in connection with the enclosed Figuresexplains the invention in more detail with reference to embodiments.

FIG. 1 shows a schematic illustration of a device for analyzing a samplecontaining fluorophores according to a first embodiment of theinvention.

FIG. 2 shows a diagram with the curves of the transmission properties ofthe filter, the excitation efficiency of the reference object, theexcitation efficiency of the fluorophore and the emission spectrum ofthe fluorophore as a function of the wavelength in the first embodimentof the invention.

FIG. 3 shows a diagram with the curves of the excitation efficiency ofthe fluorophore and the resulting excitation efficiency of the referenceobject changed by the filter in the first embodiment of the invention.

FIG. 4 shows a schematic illustration of a device for analyzing a samplecontaining fluorophores according to a second embodiment of theinvention.

FIG. 5 shows a diagram with the curves of the transmission properties ofthe filter, the excitation efficiency of the reference object, theexcitation efficiency of the fluorophore and the emission spectrum ofthe fluorophore as a function of the wavelength in the second embodimentof the invention.

FIG. 6 shows a diagram with the curves of the excitation efficiency ofthe fluorophore and the resulting excitation efficiency of the referenceobject changed by the filter in the second embodiment of the invention.

FIG. 7 shows a schematic illustration of a device for analyzing a samplecontaining fluorophores according to a third embodiment of theinvention.

FIG. 8 shows a diagram with the curves of the transmission properties ofthe filter, the excitation efficiency of the reference object, theexcitation efficiency of the fluorophore and the emission spectrum ofthe fluorophore as a function of the wavelength in a third embodiment ofthe invention.

FIG. 9 shows a diagram with the curves of the excitation efficiency ofthe fluorophore and the resulting excitation efficiency of the referenceobject changed by the filter in the third embodiment of the invention.

FIG. 10 shows a schematic illustration of a device for analyzing asample containing fluorophores according to a fourth embodiment of theinvention.

FIG. 11 shows a diagram with the curves of the transmission propertiesof the filter, the excitation efficiency of the reference object, theexcitation efficiency of the fluorophore and the radiation spectrum ofthe fluorophore as a function of the wavelength in the fourth embodimentof the invention.

FIG. 12 shows a diagram with the curves of the excitation efficiency ofthe fluorophore and the resulting excitation efficiency of the referenceobject changed by the filter in the fourth embodiment of the invention.

FIG. 13 shows a schematic illustration of a device for analyzing asample containing fluorophores according to a fifth embodiment of theinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 shows a schematic illustration of a device 10 for analyzing asample 16 containing fluorophores according to a first embodiment of theinvention. The device 10 comprises a light source 12 which emits lighthaving a wavelength λ_(ex) onto an internal reference standard 14 whichserves as a reference object and onto the sample 16 supplied to thedevice 10 in the form of a test strip, as schematically illustrated bythe arrows P1 and P2. The light emitted from the internal referencestandard 14 has a wavelength λ_(em1) and, as indicated by the arrow P3,is incident on a detection area of a detector 18. The light emitted fromthe sample 16 and having a wavelength λ_(em2) is incident on thedetection area of the detector 18, as indicated by the arrow P4. Thelight source 12 sequentially emits light onto the reference standard 14and the sample 16. A filter 20 arranged between the light source 12 andthe internal reference standard 14 causes that the different spectralcurves of the excitation efficiency of the internal reference standard14 and of the spectral sensitivity of the fluorophores of the sample 16given a change in the wavelength λ_(ex) of the light emitted from thelight source 12 adapt to each other. As a result thereof, in the case ofa change in wavelength of the light radiation generated by the lightsource 12, the analysis result of the device 10 is not or only slightlyinfluenced. In the embodiment of the invention, the reference standard14 contains fluorophores and is also referred to as fluorescencestandard.

In FIG. 2, a diagram with the spectral curves of the excitationefficiency 22 of the fluorophore, of the emission spectrum 24 of thefluorophore, of the transmission characteristic 26 of the filter 20 andof the excitation efficiency 28 of the internal reference standard 14 isillustrated. Here, the wavelength λ_(ex1) is the nominal wavelength ofthe light source 12, which in the present embodiment is 638 nm, and thewavelength λ_(ex1) is the change in wavelength of the light source 12caused due to the aging of components of the light source 12 and/or dueto environmental influences, such as a change in temperature. In thepresent embodiment, the wavelength λ_(ex2) is, for example, 642 nm. Whenthe wavelength of the light emitted from the light source 12 changesfrom 638 nm to 642 nm, the excitation efficiency of the fluorophorecontained in the sample 16 increases from the factor 0.44 to the factor0.51 and thus from 44% to 51%. However, given a change in the wavelengthλ_(ex) from 638 nm to 642 nm, the excitation efficiency 28 of thereference object decreases from 0.89 to 0.85 and thus from 89% to 85%.In the case of a quantitative determination of the fluorophores in thesample 16 on the basis of a comparison of the measurement value detectedby the detector 18 when the sample 16 is irradiated and the measurementvalue detected by the detector 18 when the internal reference standard14 is irradiated, this results in a considerable evaluation error in theevaluation of the measurement values. In the present embodiment, thefilter 20 causes, at least in the range between the wavelength λ_(ex1)and λ_(ex2), a coincidence of the resulting excitation efficiency of theinternal reference standard 14 caused by the filter 20. The spectralcurve of the resulting excitation efficiency of the internal referencestandard 14 caused by the filter 20 is shown in FIG. 3 by the curve 30.The curve 30 results from the product of the excitation efficiency 28and the transmission characteristic 26 of the filter 20, as shown inFIG. 2.

As shown in FIG. 3, the excitation efficiency 22 of the fluorophores ofthe sample and the curve 30 of the resulting excitation efficiency ofthe internal reference standard 14 change in the range between the firstwavelength λ_(ex1) and the second wavelength λ_(ex2) in the same mannerso that as a result thereof, given a change in wavelength in this rangebetween λ_(ex1) and λ_(ex2) resulting excitation efficiency 30 as wellas the excitation efficiency 22 are changed in the same manner so thatgiven a relative determination of the quantity of the fluorophores inthe sample 16 this sample 16 is directly evaluated and measurementerrors of the device 10 can be avoided or, respectively, compensated forby the filter 20.

In FIG. 4, a schematic illustration of a device 40 for analyzing thesample 16 containing fluorophores according to a second embodiment ofthe invention is shown. Elements having the same structure or the samefunction are identified with the same reference signs.

In contrast to the first embodiment according to FIG. 1, instead of thefilter 20 arranged between the light source 12 and the internalreference standard 14, in the second embodiment of the invention afilter 42 is provided that is arranged between the light source 12 andthe sample 16. The spectral curve of the transmission characteristic ofthe filter 42 is illustrated in FIG. 5 and identified with the referencesign 44. Further, the internal reference standard 46 used in the secondembodiment has an excitation efficiency deviating from the internalreference standard 14, and whose curve is identified with the referencesign 48 in FIG. 5. However, the reference standard 46, too, containsfluorophores. By the filter 42, the spectral curve of the resultingexcitation efficiency is changed as a result of the combination of thefilter 42 and the sample 16 and it extends in the relevant wavelengthrange between the wavelength λ_(ex1) and λ_(ex2) parallel to theexcitation efficiency 48 of the reference standard 46, as shown by thecurve 50 in FIG. 6.

In FIG. 7, a schematic illustration of a device 60 for analyzing asample 16 containing fluorophores according to a third embodiment of theinvention is shown. The device 60 comprises a monitor diode 62. Thelight source 12 serves as an excitation light source and emitsexcitation light both onto a monitor diode 62 for monitoring theintensity of the light radiation generated by the light source 12 andonto the sample 16. The light emitted from the sample 16 is incident onthe detector element 18, wherein a filter 64 is arranged between thesample 16 and the detector element 18 to filter out the light of thelight source 12 that is reflected on the sample 16 and to only supplythe fluorescence light emitted from the fluorophores contained in thesample 16 to the detection area of the detector 18. Likewise, in theembodiments according to FIG. 1 and according to FIG. 4, a filter can beprovided in the path P4 for filtering out the excitation light emittedfrom the light source 12 and reflected by the sample 16. The device 60comprises a second inventive filter 66 which is arranged between thelight source 12 and the monitor diode 62 and whose transmissioncharacteristic is illustrated in FIG. 8 by the curve 68. The detectionsensitivity of the monitor diode 66 is illustrated in FIG. 8 by thecurve 70. By the combination of the transmission characteristic 68 ofthe filter 66 and the detection characteristic 70 of the monitor diode62 the resulting transmission characteristic 72 of the combination offilter 66 and monitor diode 62 as illustrated in FIG. 9 results. In therange between the wavelengths λ_(ex1) and λ_(ex2), the curves of theresulting detection sensitivity 72 and the excitation efficiency 22coincide so that as a result thereof, effects of a change in wavelengthof the excitation light of the excitation light source 12 from λ_(ex1)to λ_(ex2) or, respectively, of a wavelength in the range betweenλ_(ex1) and λ_(ex2) has no such effect that the change in wavelengthdistorts the measurement result of the device 60.

The device 60 further comprises a reference light source 13 which emitslight of a reference light wavelength onto the reference standard 68 andthe monitor diode 62. The excitation light source 12 and the referencelight source 13 are operated alternatively, so that the light emitteddue to the reference light from the reference standard 68, i.e.fluorescence light and/or reflected light, is detected by the detectorunit 18, the light emitted from the reference light source 13 beingsimultaneously detected by the monitor diode 62. In the presentembodiment, the reference light is passed through the filter 66, whereinin the wavelength range of the reference light emitted from thereference light source 13 the filter 66 has a substantially lowerinfluence on the reference light than in the wavelength range of theexcitation light of the excitation light source 12. Preferably, thereference light of the reference light source 13 has a wavelength whichlies in the wavelength spectrum of the fluorescence light emitted fromthe sample 16 and does not lie in the wavelength range of the excitationlight.

In FIG. 10, a schematic illustration of a device 80 for analyzing asample 16 containing fluorophores according to a fourth embodiment ofthe invention is illustrated.

In contrast to the device 60 according to FIG. 7, instead of the filter66 between the excitation light source 12 and the monitor diode 62 (FIG.7) a filter 82 is arranged between the light source 12 and the sample16. The curve of the transmission characteristic of the filter 82 isidentified with the reference sign 84 in FIG. 11. The detectionsensitivity of the monitor diode 86 is lower than the detectionsensitivity of the monitor diode 62 according to FIG. 7 and isillustrated in FIG. 11 by the curve 88. By the filter characteristic 84of the filter 82, there results the resulting excitation efficiency 90illustrated in FIG. 12, which results from the combination of the curveof the excitation efficiency 22 of the fluorophores of the sample 16 andthe filter characteristic 84 of the filter 82. In the range between thewavelength λ_(ex1) and λ_(ex2), then the resulting excitation efficiency90 extends parallel to the spectral detection sensitivity 88 of themonitor diode 86 so that as a result thereof measurement errors due tothe change in wavelength of the light source in the range between thewavelengths λ_(ex1) and _(λex2) can be reduced or completely removed.

In FIG. 13, a schematic illustration of a device 100 for analyzing asample 16 containing fluorophores according to a fifth embodiment of theinvention is shown. In contrast to the device 80 according to FIG. 10, afurther filter 102 is provided between the reference light source 13 andthe reference standard 68. By means of the filter 102, the spectralcharacteristic of the reference standard 68 can be adapted to thespectral curve of the detection sensitivity of the monitor diode 86.

Likewise, the filter 66 in the third embodiment of the inventionaccording to FIG. 7 can have such a fourth characteristic that it adaptsboth the spectral detection sensitivity of the monitor diode 62 to thespectral curve of the excitation efficiency of the fluorophores of thesample 16 and to the excitation efficiency or, respectively, theemission efficiency of the reference element 68. The reference element68 is also referred to as reference object, serves as a referencestandard and can be designed as an optical element.

Further, the filter 66 and the monitor diode 62 can be arranged withinone unit, in particular the filter 66 can be designed as a coating of asurface of the detection area of the monitor diode 62. Also, thereference standard 68 can be combined in an arrangement with the filter102, in particular the filter 102 can be applied as a coating on thereference standard 68.

In particular, the ratio of the energy supplied to the reference objectvia the immitted light to the energy emitted from the reference objectis regarded as emission efficiency. Here, the energy which is emittedfrom the reference object and is relevant for the emission efficiencycan be caused by a reflection of a part of the irradiated light, by atransmission of the emitted light or by an excitation of a substancecontained in the reference object for emitting a radiation. Such asubstance can in particular comprise a suitable fluorophore.

In all embodiments of the invention, the spectral curves of referencestandard and fluorophore as well as between monitor diode and sample areadapted to each other in the relevant wavelength range so that theeffect on a measurement result due to the changes in wavelength can beminimized or completely compensated for. Embodiments of the inventionare also possible in which the device has both a reference object and asample as well as a monitor diode, wherein then the detectionsensitivity of the monitor diode, the excitation efficiency of thefluorophore and the excitation efficiency of the reference object areadapted to each other and have a parallel curve at least in a relevantwavelength range in which variations of the wavelength of the lightemitted from the light source 12 might occur. Instead of a filterbetween the light source and the reference object or between the lightsource and the sample 16, each time a filter can be arranged between thelight source and the reference element and the light source and thesample to adapt the curves of the excitation efficiencies of thefluorophores of the sample and of the fluorophores of the referenceobject to each other. Further, both a first filter between the lightsource and the monitor diode and a second filter between the lightsource and the sample can be provided to adapt the detection sensitivityof the monitor diode and the excitation efficiency of the sample in therelevant spectral range between λ_(ex1) and λ_(ex2).

1. A device for determining the concentration of fluorophores in a sample, with a light source (12) for emitting light onto the sample (16) and for emitting light onto a fluorescence standard (14, 46), wherein the fluorophores of the sample (16), given an immission of light of a first wavelength (λ_(ex1)) have a first excitation efficiency and, given an immission of light of a second wavelength (λ_(ex2)), have a second excitation efficiency, (λ_(ex2)), wherein the fluorophores of the fluorescence standard (14, 46), given an immission of light of the first wavelength (λ_(ex1)) have a third excitation efficiency and, given an immission of light of the second wavelength (λ_(ex2)) onto the reference object (14, 46), have a fourth excitation efficiency, with an optical element (20, 42) which is arranged between the light source (12) and the sample (16) and/or between the light source (12) and the fluorescence standard (14, 46) and/or as an optically effective layer on the fluorescence standard (14) and which, due to its optical property, adapts a first difference between the first excitation efficiency and the second excitation efficiency and a second difference between the third excitation efficiency and the fourth excitation efficiency to each other.
 2. A device for determining the concentration of fluorophores in a sample, with a reference light source (13) for emitting reference light onto an optical reference object and for emitting reference light onto a monitor diode (62, 86), wherein the optical reference object couples in a part of the reference light emitted from the reference light source (13) in the direction of a detection element (18), with an excitation light source (12) for emitting excitation light onto the fluorophores of the sample (16) and for emitting excitation light onto the monitor diode (62, 86), wherein the fluorophores of the sample (16) emit fluorescence light in the direction of the detection element (18) given an immission of excitation light, wherein the fluorophores, given an immission of excitation light of a first wavelength (λ_(ex1)) have a first excitation efficiency and, given an immission of excitation light of a second wavelength (λ_(ex2)), have a second excitation efficiency, wherein the monitor diode (62, 86), given an immission of excitation light of a first wavelength (λ_(ex1)) has a first detection sensitivity and, given an immission of excitation light of a second wavelength (λ_(ex2)) onto the monitor diode (62, 86), has a second detection sensitivity, with an optical element (66, 82) which is arranged between the excitation light source and the sample and/or between the excitation light source and the monitor diode (62, 86) and/or as an optically effective layer on the monitor diode (62, 86) and which, due to its optical property, adapts a first difference between the first excitation efficiency and the second excitation efficiency and a second difference between the first detection sensitivity and the second detection sensitivity to each other.
 3. The device according to claim 1, characterized in that the device has a detection element (18) for detecting the light emitted from the sample (16) given an immission of light of the light source (12) onto the sample (16) and/or for detecting the light emitted from the reference object (14, 46) given an emission of light of the light source (12) onto the reference object (14, 46).
 4. The device according to claim 3, characterized in that the detection element (18), when detecting the light emitted from the sample (16) given an immission of light of the light source (12) onto the sample (16), determines a detection value, by means of which a control unit determines the quantity of the fluorophores in the sample (16) and/or the concentration of the substance in the sample (16) containing the fluorophores.
 5. The device according to claim 1, characterized in that the optical property of the optical element (20, 42, 66, 82) influences the light emitted from the sample (16) given the immission of light of the light source (12) and/or the light emitted from the fluorescence standard (14, 46) and/or the light emitted from the reference object such that the first difference is approximated to the second difference, preferably that the second difference is equal to the first difference.
 6. The device according to claim 1, characterized in that the optical element (20, 42, 66, 82) adapts the spectral curve of the excitation efficiency of the sample (16) and the spectral curve of the excitation efficiency of the fluorescence standard (14, 46) at least in the wavelength range between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)), and/or in that the optical element (20, 46, 66, 82) adapts the spectral curve of the excitation efficiency of the sample (16) and the spectral curve of the detection sensitivity of the monitor diode (62, 86) at least in the wavelength range between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)).
 7. The device according to claim 6, characterized in that the optical element (20, 42, 66, 82) adapts the spectral curve of the excitation efficiency of the sample (16) and the spectral curve of the excitation efficiency of the fluorescence standard (14, 46) at least in the wavelength range between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)) such that they coincide with one another at least in this wavelength range, and/or in that the optical element (20, 42, 66, 82) adapts the spectral curve of the excitation efficiency of the sample (16) and the spectral curve of the detection sensitivity of the monitor diode (62, 86) at least in the wavelength range between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)).
 8. The device according to claim 1, characterized in that the optical element is a filter and in that the optical property of the optical element is the transmission property of the filter.
 9. The device according to claim 1, characterized in that the first wavelength (λ_(ex1)) is the nominal wavelength of the light source (12) or, respectively, of the excitation light source (12) and in that the second wavelength (λ_(ex2)) is a wavelength different from the first wavelength, which in particular results due to temperatures influences and/or aging of at least one component of the light source (12) or, respectively, of the excitation light source (12).
 10. The device according to claim 9, characterized in that the difference between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)) is in the range between 0.1 nm to 5 nm.
 11. The device according to claim 1, characterized in that the light source (12), the excitation light source (12) and/or the reference light source (13) comprise a laser light source, an LED light source or a white light source with a filter arrangement for generating light in a narrow-band range, wherein the first wavelength (λ_(ex1)) lies within the narrow-band range, preferably in the middle thereof.
 12. The device according to claim 1, characterized in that the excitation light source (12) emits light simultaneously onto the sample (16) and onto the fluorescence standard (14, 46) or in that the light source (12) emits light sequentially onto the sample (16) and onto the fluorescence standard (14, 46) and/or in that a control unit sequentially activates the excitation light source (12) and the reference light source (13).
 13. The device according to claim 1, characterized in that the reference object (14, 46) serves as an internal reference standard, as a calibration standard and/or as a quality assurance standard.
 14. The device according to claim 1, characterized in that the reference object (14, 46) comprises a fluorescence standard and/or an optical element serving as a reference standard.
 15. A method for determining the concentration of fluorophores in a sample, in which by means of a light source (12) light is emitted onto a substance of the sample (16) containing fluorophores and light is emitted onto a fluorescence standard (14, 46), wherein the fluorophores in the sample (16), given an immission of light of a first wavelength (λ_(ex1)), have a first excitation efficiency and, given an immission of light of a second wavelength (λ_(ex2)) onto the fluorophores, have a second excitation efficiency, wherein the fluorophores of the fluorescence standard (14, 46), given the immission of light of a first wavelength (λ_(ex1)), have a third excitation efficiency and, given an immission of light of the second wavelength (λ_(ex2)) onto the fluorescence standard (14, 46), have a second excitation efficiency, and in which by means of an optical element (20, 42) arranged between the light source (12) and the sample (16) and/or between the light source (12) and the fluorescence standard (14, 46) a first difference between the first excitation efficiency and the second excitation efficiency and a second difference between the third excitation efficiency and the fourth excitation efficiency are adapted to each other due to the optical property of the optical element.
 16. The device according to claim 2, characterized in that the device has a detection element (18) for detecting the light emitted from the sample (16) given an immission of light of the light source (12) onto the sample (16) and/or for detecting the light emitted from the reference object (14, 46) given an emission of light of the light source (12) onto the reference object (14, 46).
 17. The device according to claim 2, characterized in that the optical property of the optical element (20, 42, 66, 82) influences the light emitted from the sample (16) given the immission of light of the light source (12) and/or the light emitted from the fluorescence standard (14, 46) and/or the light emitted from the reference object such that the first difference is approximated to the second difference, preferably that the second difference is equal to the first difference.
 18. The device according to claim 2, characterized in that the optical element (20, 42, 66, 82) adapts the spectral curve of the excitation efficiency of the sample (16) and the spectral curve of the excitation efficiency of the fluorescence standard (14, 46) at least in the wavelength range between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)), and/or in that the optical element (20, 46, 66, 82) adapts the spectral curve of the excitation efficiency of the sample (16) and the spectral curve of the detection sensitivity of the monitor diode (62, 86) at least in the wavelength range between the first wavelength (λ_(ex1)) and the second wavelength (λ_(ex2)).
 19. The device according to claim 2, characterized in that the optical element is a filter and in that the optical property of the optical element is the transmission property of the filter.
 20. The device according to claim 2, characterized in that the first wavelength (λ_(ex1)) is the nominal wavelength of the light source (12) or, respectively, of the excitation light source (12) and in that the second wavelength (λ_(ex2)) is a wavelength different from the first wavelength, which in particular results due to temperatures influences and/or aging of at least one component of the light source (12) or, respectively, of the excitation light source (12).
 21. The device according to claim 2, characterized in that the light source (12), the excitation light source (12) and/or the reference light source (13) comprise a laser light source, an LED light source or a white light source with a filter arrangement for generating light in a narrow-band range, wherein the first wavelength (λ_(ex1)) lies within the narrow-band range, preferably in the middle thereof.
 22. The device according to claim 2, characterized in that the excitation light source (12) emits light simultaneously onto the sample (16) and onto the fluorescence standard (14, 46) or in that the light source (12) emits light sequentially onto the sample (16) and onto the fluorescence standard (14, 46) and/or in that a control unit sequentially activates the excitation light source (12) and the reference light source (13).
 23. The device according to claim 2, characterized in that the reference object (14, 46) serves as an internal reference standard, as a calibration standard and/or as a quality assurance standard.
 24. The device according to claim 2, characterized in that the reference object (14, 46) comprises a fluorescence standard and/or an optical element serving as a reference standard. 